Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer’s disease-like pathology in hippocampal neuronal cells via the blocking of Ca2+/Calpain/Cdk5-mediated mitochondrial fragmentation

Oxidative stress plays an essential role in the progression of Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder. Streptozotocin (STZ)-induced abnormal brain insulin signaling and oxidative stress play crucial roles in the progression of Alzheimer’s disease (AD)-like pathology. Peroxiredoxins (Prxs) are associated with protection from neuronal death induced by oxidative stress. However, the molecular mechanisms underlying Prxs on STZ-induced progression of AD in the hippocampal neurons are not yet fully understood. Here, we evaluated whether Peroxiredoxin 1 (Prx1) affects STZ-induced AD-like pathology and cellular toxicity. Prx1 expression was increased by STZ treatment in the hippocampus cell line, HT-22 cells. We evaluated whether Prx1 affects STZ-induced HT-22 cells using overexpression. Prx1 successfully protected the forms of STZ-induced AD-like pathology, such as neuronal apoptosis, synaptic loss, and tau phosphorylation. Moreover, Prx1 suppressed the STZ-induced increase of mitochondrial dysfunction and fragmentation by down-regulating Drp1 phosphorylation and mitochondrial location. Prx1 plays a role in an upstream signal pathway of Drp1 phosphorylation, cyclin-dependent kinase 5 (Cdk5) by inhibiting the STZ-induced conversion of p35 to p25. We found that STZ-induced of intracellular Ca2+ accumulation was an important modulator of AD-like pathology progression by regulating Ca2+-mediated Calpain activation, and Prx1 down-regulated STZ-induced intracellular Ca2+ accumulation and Ca2+-mediated Calpain activation. Finally, we identified that Prx1 antioxidant capacity affected Ca2+/Calpain/Cdk5-mediated AD-like pathology progress. Therefore, these findings demonstrated that Prx1 is a key factor in STZ-induced hippocampal neuronal death through inhibition of Ca2+/Calpain/Cdk5-mediated mitochondrial dysfunction by protecting against oxidative stress.


Plasmid construction preparation of stable cell line
Prx1 gene including plasmid (pLenti6.3-Prx1-V5;Thermo Fisher Scientific) kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea).Prx1 was amplified by PCR with LA Taq polymerase (TaKaRa, Shiga, Japan).These genes were cloned into a gateway entry vector pCR8/GW/TOPO (Thermo Fisher Scientific) to generate expression clones by performing LR recombination between the entry vector and gateway destination vectors pLenti6.3/V5-DEST(Thermo Fisher Scientific).pLenti6.3/V5-DESTvector has a C-terminal V5 epitope that aids in detecting recombinant proteins during immunoblotting analysis 51 .The sequences of the constructed vectors were confirmed by performing restriction mapping and DNA sequencing.1 μg of pLenti6.3-Prx1-V5plasmid was transfected into the HT-22 cells by using effectene (Qiagen, Hilden, Germany), according to the manufacturer's instructions.After 24 h, the transfected cells were selected using 8 μg/mL blasticidin (Thermo Scientific).

MTT assay
Mock-and Prx1-expressed HT-22 cells were cultured on 96-well plates for 24 h.After experiments, each cell sample was incubated for 30 min at 37 °C with MTT (0.5 mg/mL; Sigma-Aldrich), and then 100 μL DMSO (Sigma-Aldrich) was added.Absorbance was measured at 550 nm.

Measurements of intracellular ATP levels
The intracellular ATP levels were determined using an ATP determination kit (Thermo Fisher Scientific) following the manufacturer's instructions.After experiments, whole protein lysates at a concentration of 1 μg/mL were used for the measurements of intracellular ATP levels.

Mitochondrial imaging
To observe mitochondrial morphology, DsRed2-mito including plasmid (pLenti6.3-DsRed2-mito)was kindly provided by Dr. Dong-Seok Lee (Kyungpook National University, Daegu, Korea).DsRed2-mito expressed HT-22 cells were seeded on 0.01% poly-D-lysine-coated round coverslips and were incubated for 24 h.After experiments, cells were washed with PBS, and fixed with 4% paraformaldehyde for 1 h.After washing, the coverslips were mounted on slides with mounting medium (VECTOR Laboratories, CA, USA).To observe mitochondrial morphology in Prx1-expressed HT-22 cells, MitoTracker Green (100 µM; Thermo Fisher Scientific) was incubated with the cells.Mitochondrial images were acquired using the LSM-710 confocal microscope (Carl Zeiss, Jena, Germany).Mitochondrial length measurements were performed using Image J software as previously described 52 .Briefly, average mitochondrial length was evaluated from more than 30 mitochondria particles per cell in over 10 cells.

Determination of intracellular Ca 2+ levels
Intracellular Ca 2+ level was measured using Fluo-4 AM (Thermo Fisher Scientific).After experiment, cells were incubated with Fluo-4 AM for 30 min at 37 °C.After washing with HBSS, images of Fluo-4 AM were obtained using an ECLIPSE Ti-U microscope (Nikon, Tokyo, Japan), and the fluorescence intensity of Fluo-4 AM was measured with image J software.

Measurements of intracellular ROS
Intracellular ROS generation was assessed using CM-H 2 DCFDA.After experiments, cells were incubated with 5 μM CM-H 2 DCFDA (Thermo Fisher Scientific) for 30 min at 37 °C, and then analyzed using an ECLIPSE Ti-U microscope (Nikon) and FACSCalibur flow cytometry (BD Biosciences).

Statistical analysis
The data represent the mean ± SD from three independent experiments (n ≥ 3).Experimental differences were tested for statistical significance using GraphPad Prism 9 software (San Diego, CA, USA).Multiple group analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for normally distributed datasets.Statistical significance was set at p < 0.05, and is indicated on the graphs using asterisks; P-values < 0.01 and < 0.001 were indicated by two and three asterisks, respectively.

STZ-mediated Prx1 induction inhibits the progression of AD-like pathology
We investigated the Prx1 protein expression levels in HT-22 cells following time-dependent STZ treatments.STZ concentration (10 mM) referred to induce AD-like pathology in HT-22 cells 53 .We found that Prx1 protein expression level was gradually increased after 12 h STZ treatments (Fig. 1A).To assess the effect of inducible Prx1 expression by STZ on the process of AD-like pathology, we generated V5-tagged Prx1 (Prx1-V5) stably expressing HT-22 cells line by using transfection of pLtneti6.3-Prx1-V5.We identified the exogenous expression of Prx1 (Prx1-V5) by western blotting using Prx1 and V5-tag antibodies (Fig. 1B).Prx1 expression reversed STZ-induced decreased cell viability and increased apoptotic markers, such as cleaved caspase-3 and cleaved PARP (Fig. 1C,D).Furthermore, we determined the effect of Prx1 on STZ-induced neuronal loss and synaptic function using immunoblotting with NeuN (neuronal marker), and PSD95 (post-synapse marker).Our results showed that STZ-induced down-regulation of NeuN and PSD95 were inhibited in Prx1 expression (Fig. 1E,F).We also confirmed that Prx1 impacted the phosphorylation of tau epitopes, such as p-Tau(S262) and AT8(S202/ T205).Up-regulated p-Tau (S262) and AT8(S202/T205) by STZ treatment were also suppressed by Prx1 expression (Fig. 1G).These results suggested that Prx1 induction by STZ regulates the STZ-induced process of AD-like pathology, apoptotic neuronal loss, synaptic loss, and tauopathy in hippocampal cell lines.

Prx1 prevents STZ-induced mitochondrial fragmentation and dysfunction
We previously reported that changes in mitochondrial morphology and mitochondrial function relate to STZinduced progression of AD-like pathology 53 .Therefore, we measured changes in intracellular ATP levels after STZ treatment for 12 h in mock-and Prx1-expressed HT-22 cells.Our results indicated that the down-regulated intracellular ATP level by STZ treatment was suppressed by Prx1 expression (Fig. 2A).We determined whether Prx1 affects STZ-induced mitochondrial fragmentation.To observe changes in mitochondrial morphology after STZ treatment for 12 h in HT-22 and Prx1-expressed HT-22 cells, we stained with mitotracker green after experiments.Our result indicated that the decrease of mitochondrial average length by STZ treatment in HT-22 cells was inhibited in Prx1-expressed HT-22 cells (Fig. 2B,C).STZ-induced mitochondrial fragmentation depended on the Drp1 translocation from the cytoplasm to the mitochondria by phosphorylation of Drp1(S616) via activating Cdk5/p25 signaling pathway 53 .Therefore, we assess the effects of Prx1 on the Cdk5/p25-mediated Drp1 activation by STZ treatment for 12 h.Our results indicated that the STZ-induced increase in the level of mitochondrial Drp1 was inhibited by Prx1 expression (Fig. 2C).Drp1(S616) phosphorylation was increased by STZ treatment for 12 h, and up-regulated Drp1(S616) phosphorylation was repressed by Prx1 expression (Fig. 2D).Moreover, increased level of p25 by STZ treatment for 6 h, which is known to show the highest level of p25 by STZ treatment in HT-22 cells 53 , was inhibited by Prx1 expression (Fig. 2E).Therefore, we demonstrate that induction of Prx1 by STZ regulates Cdk5-mediated mitochondrial fragmentation and dysfunction by inhibiting cleavage of p35 to p25.

Prx1 regulates STZ-induce Ca 2+ -mediated mitochondrial fragmentation and dysfunction through calpain/Cdk5-mediated Drp1 activation
Previous reports indicated that Prx5 inhibits the accumulation of intracellular Ca 2+ , calpain activation, and Cdk5 activation in an amyloid-beta (Aβ) oligomer-mediated AD cellular model 54 .Therefore, we measured intracellular Ca 2+ levels using the intracellular Ca 2+ indicator Fluo-4 AM, in time-dependent STZ-treated HT-22 cells.Intracellular Ca 2+ was measured from 3 h STZ treatment and maintained to 6 h (Fig. 3A).An increased level of STZ-induced intracellular Ca 2+ was down-regulated by Prx1 expression (Fig. 3B).The elevated expression level of calpain-2 is known to reflect calpain activation in neuron cells 40,41 .Thus, we then assessed whether Prx1 affected calpain-2 expression.We found that calpain-2 expression level was the highest at 1.5 h STZ treatment time, and was gradually down-regulated to 6 h (Fig. 3C).Prx1 inhibited STZ-induced increase of calpain-2 expression (Fig. 3D).We then assessed whether Prx1 influenced STZ-induced mitochondrial fragmentation through intracellular Ca 2+ regulation using an intracellular Ca 2+ chelator, BAPTA-AM.We found that inhibition of STZ-induced Ca 2+ accumulation prevented the calpain activation and Cdk5 activation by p35 cleavage to p25 (Fig. 4A,B).Furthermore, STZ-induced increases of fragmented mitochondria and mitochondrial dysfunction were restored by intracellular Ca 2+ chelation via controlling Drp1 activation (Fig. 4C,D,E,F).These results suggest that Prx1 regulates calpain/Cdk5-mediated mitochondrial fragmentation by controlling STZ-induced intracellular Ca 2+ accumulation.

Effect of intracellular Ca 2+ on the STZ-induced AD-like pathology
Past demonstrations indicated that intracellular Ca 2+ or control of mitochondrial morphology were key mediators in AD 54 .As we proved the role of Ca 2+ as a regulator of STZ-induced mitochondrial fragmentation, we determined the effect of STZ-induced Ca 2+ on the progression of AD-like pathology.STZ-induced Ca 2+ inhibition with BAPTA-AM suppressed increased neuronal apoptosis and neuronal loss.The increased level of cleaved caspase-3 and cleaved PARP, reduced the level of NeuN by STZ reversed by BAPTA-AM treatment (Fig. 5A,B).In addition, the STZ-induced increased synaptic loss and Tau activation were confirmed with PSD95, p-Tau(S262), and AT8(S202/T205).STZ-induced decrease of PSD95 and increase of p-Tau(S262) and AT8(S202/T205) were attenuated by BAPTA-AM treatment (Fig. 5C,D).These results indicated that STZ-mediated accumulation of intracellular Ca 2+ can influence the progression of STZ-induced AD-like pathology.

Influence of ROS on STZ-induced AD-like pathology via Ca 2+ /calpain/Cdk5-mediated mitochondrial fragmentation
Previous reports suggested that oxidative stress relates to the accumulation of intracellular Ca 2+ -mediated calpain activation, and Prx blocks the increase in intracellular Ca 2+ accumulation 55,56 .Therefore, to assess whether Prx1 affected Ca 2+ -mediated calpain activation using Prx1 antioxidant capacity, we inhibited STZ-induced ROS production using antioxidant molecule, N-acetyl-cysteine (NAC).We first determined the effect of Prx1 on STZ-induced intracellular ROS level in STZ-induced HT-22 cells and Prx1-expressed HT-22 cells with CM-H 2 DCFDA.Our results indicated that increased levels of intracellular ROS induced by STZ treatment at 12 h were suppressed by Prx1 expression (Fig. 6A,B).We investigated the impact of STZ-induced ROS inhibition on calpain activation and p35 cleavage.Up-regulation of calpain-2 and p25 protein levels was restored by NAC treatment (Figure C and D).Increase of mitochondrial fragmentation and dysfunction, a downstream pathway of Ca 2+ /calpain/Cdk5 was rescued by ROS scavenge through inhibition of Drp1(S616) phosphorylation and mitochondrial location (Fig. 6E,F,G,H).We then assessed the effect of STZ-induced ROS inhibition on the STZinduced progression of AD-like pathology.Our results showed that STZ-induced increases in neuronal apoptosis, neuronal loss, synaptic loss, and Tau phosphorylation were prevented by NAC treatment (Fig. 7).These results suggested that STZ-induced progression of AD-like pathology in HT-22 cells was involved in increased ROS levels through governing Ca 2+ /Calpain/Cdk5-mediated mitochondrial fragmentation.

Discussion
In our previous research, we developed an STZ-induced effective and clinically relevant AD-like model in non-human primates and rodents through intra-cisternal magna (ICM) route, characterized by cerebral and hippocampal damage, disintegration of the neurovascular unit, Aβ deposition, neuroinflammation, and Cdk5 activation [57][58][59][60] .Recently, we suggested that regulation of Cdk5/Drp1-dependent mitochondrial morphology plays a role in the potential inhibitor of abnormal metabolic functions associated with AD-like pathogenesis 53 .Based on these findings, a more detailed molecular mechanistic investigation of the STZ-induced AD-like pathologies is warranted.
Oxidative stress precedes the onset of significant AD pathology in the brains of patients and animal models with AD 61,62 .Antioxidant defense systems in the response to oxidative stress, similar to changes in the expression of Prx subtypes seem to be involved in AD pathology, but this remains controversial [63][64][65][66] .Mounting evidence determined that various types of Prxs, such as Prx5 and Prx6 have been associated with regulation of the progress of AD pathologies 33,54,67 .Specifically, Prx1 was mainly expressed in oligodendrocytes and astrocytes and was detected in a few neuronal cells 68,69 .However, Prx1 expression was increased in an Aβ-resistant neuronal cell line response to oxidative stress 63 .Therefore, we focused on the role of Prx1 to assess the association with an STZ-mediated antioxidant response in HT-22 hippocampus cell line.We found that Prx1 was upregulated in a time-dependent manner in response to STZ-mediated oxidative stress.However, it was confirmed that although the expression of Prx1 increased by twofold 24 h after STZ treatment, it was insufficient to prevent the progression of AD-like pathology and cell death.Therefore, to find out the role of Prx1 in increasing by STZ treatment, we produced the Prx1 overexpression cell line in HT-22 cells to assume a situation in which Prx1 was expressed at an early timepoint of STZ treatment.
Prx1 overexpression inhibited STZ-mediated neuronal apoptosis, synaptic loss, and tau phosphorylation by preventing cellular ROS accumulation.Furthermore, STZ-mediated mitochondrial fragmentation was suppressed by Prx1 overexpression through the prevention of Cdk5-dependent Drp1 phosphorylation.Our results suggested that Prx1 suppressed neuronal apoptosis by inhibiting the increase of cleaved caspase-3.Previous research demonstrated that caspase-3 activation was regulated by mitochondrial morphology in conditions of neuronal death [70][71][72] .Furthermore, amount studies found that Prx1 was a key inhibitor of the caspase-3 activation in neuronal death states 73,74 .It has been suggested that dysregulation of Cdk5 homeostasis has pathological relevance to AD 46,[75][76][77] .Oxidative stress is considered to be a crucial modulator of Cdk5 activation 78,79 .Prx5 was involved in Cdk5 activation by modulating oxidative stress 80,81 , and Prx1 activation inhibited Aβ-induced impaired axonal transport 82 .However, the precise relationship between Prx1 and Cdk5 activation in STZ-mediated AD-like pathogenesis are still unclear.Our findings reveal that Prx1 is an important suppressor of STZ-induced progression of AD-like pathology via Cdk5 activation and mitochondrial fragmentation.
Cdk5 activation by p25 is triggered by the activation of calpain, which is related to an accumulation of intracellular Ca 2+79,83 .Our result also showed that the elimination of STZ-induced accumulated intracellular Ca 2+ suppressed STZ-mediated calpain-2 expression, Cdk5 activation, mitochondrial fragmentation, and progression of AD-like pathology.Oxidative stress is associated with dysregulation of Ca 2+ release and signal pathway 84,85 , which in turn triggers calpain-2 activation [86][87][88] .Calpain-2 activation is known to be associated with destabilization of lysosomal membranes and the release of cathepsins in neuronal cytoplasm.Cathepsins releasing by activation of calpain-2-induced lysosomal release dysfunction has developed the calpain-cathepsin hypothesis, and it is associated with mitochondrial dysfunction and neuronal death in AD [89][90][91] .Oxidative stress is also known to induce cathepsin-mediated cell damage, and inhibition of oxidative stress showed neuroprotective effects 92 .Prx1 modulates Aβ-induced increased intracellular Ca 2+ level by inhibiting ROS accumulation 82 .Our results suggested that STZ-induced up-regulated Prx1 caused a decrease in Ca 2+ and calpain-2 levels.Apart from the antioxidant function of Prx1 that reduces peroxides via highly reactive catalytic cysteine oxidation to sulfenic acid 93 , Prx1 displays chaperone function by controlling the protein-binding partners 94 .Therefore, we proved that Prx1 decreased the STZ-induced Ca 2+ -mediated calpain-2 expression depending on Prx1 antioxidant capacity.However, it needs to study the direct role of Drp1 phosphorylation in the process of STZ-induced AD-like pathology.Our results demonstrated that the antioxidant capacity of Prx1 is a key factor in the regulation of Ca 2+ level and Ca 2+ -dependent calpain-2 expression, Cdk5-related mitochondria fragmentation.
The increased expression level of Prx1 observed in various neurodegenerative conditions 95,96 and increased expression of Prx1 contribute to resistance to oxidative stress 63 .Prx1 is known to play a protective role against ROS-mediated brain injury, such as endotoxin-induced injury 97 , Huntington's disease 98 , and acute ischemic  stroke 99 .Furthermore, microglial Prx1 participated in the protective function against endotoxin-induced proinflammatory response by regulating oxidative stress 100 , and Prx1 overexpression reduced neuronal inflammation and apoptosis by affecting microglial and astrocyte mRNA stability 95 .Therefore, these findings along with our results suggested that Prx1 plays a protective role in neurodegenerative environments by affecting neuron, astrocyte, and microglia.
Apart from these results, our findings have the limitation that it does not reflect complex environment present in the brains of AD rodents or patients because these findings were investigated from an immortalized hippocampal cell line, HT-22 cells.Furthermore, our study did not address the cause of Prx1 increase by STZ treatment.It is known that many transcription factors are involved in the induction of Prx1, and it is very important to study the mechanism of Prx1 expression by STZ treatment in the hippocampus cell line [101][102][103] .The stability of Prx1 is also one of the factors to consider.Research is needed to determine whether the reason why the expression of Prx1 increases after 12 h due to STZ treatment is due to the expression of Prx1 or changes in factors that regulate the stability of Prx1, such as ubiquitination [104][105][106] .Although several advantages of HT-22 cells [107][108][109] , such as AD pathology being well reflected or useful for molecular mechanism study, more research should be conducted in primary hippocampus cells or AD animal models based on the results of this study.Consequently, regulation of Prx1 may be a potential inhibitor of STZ-induced neurodegeneration by preventing the Ca 2+ /capain-2/Cdk5 signal pathway and may be considered as a possible strategy for developing therapies to treat the pathogenesis of AD.

Figure 2 .
Figure 2. Effect of Prx1 on STZ-induced mitochondrial dysfunction and change in mitochondrial morphology.(A) Intracellular ATP levels were measured using the ATP determination kit in STZ (12 h)-treated mock-and Prx1-expressed HT-22 cells.(B) Mitochondrial morphology was observed with mitotracker (100 μM) staining using confocal microscopy in STZ (12 h)-treated HT-22 and Prx1-expressed cells.The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm.The graph showed distribution of all mitochondrial particles and average mitochondrial length.(C) Mitochondrial location of Drp1 was analyzed by western blotting from mitochondrial isolated protein in mock-and Prx1-expressed HT-22 cells treated with STZ for 12 h.COXIV was used as the loading control for the mitochondria.(D) The protein expression levels of p-Drp1(S616) and Drp1 were determined using western blot analysis in STZ (12 h)-treated mock-and Prx1-expressed HT-22 cells.Drp1 was used as the loading control for p-Drp1(S616).(E) Protein levels of p25/35 and Cdk5 in STZ (6 h)-treated mock-and Prx1-expressed HT-22 cells were confirmed using western blotting.Blots were cropped to highlight the region of interest; full blot images in this article are provided in supplementary information.The data are presented as mean values ± SD (n ≥ 3).** denotes p < 0.01, and *** denotes p < 0.001.

Figure 4 .
Figure 4. Effect of Ca 2+ on STZ-induced calpain/Cdk5/Drp1 activation.(A) Calpain-2 protein expression level in STZ (1.5 h)-treated HT-22 cells pretreated with or without BAPTA-AM (0.5 μM) were confirmed with western blotting.(B) The proteins expression level of p25/35 and Cdk5 in STZ (6 h)-treated HT-22 cells pretreated with or without BAPTA-AM were confirmed with western blotting.(C) p-Drp1(S616), and (D) Mitochondrial Drp1 proteins expression level in STZ (12 h)-treated HT-22 cells pretreated with or without BAPTA-AM were confirmed with western blotting.COXIV was used as the loading control for the mitochondria.(E) Change in mitochondrial morphology was observed using confocal microscopy in STZ (12 h)-treated DsRed2-mito expressed HT-22 cells pretreated with or without BAPTA-AM.The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm.The graph showed distribution of all mitochondrial particles and average mitochondrial length.(F) Intracellular ATP levels were measured in STZ (24 h)-treated HT-22 cells pretreated with or without BAPTA-AM.The data are presented as mean values ± SD (n ≥ 3).** denotes p < 0.01, and *** denotes p < 0.001.

Figure 6 .
Figure 6.Effect of Prx1 antioxidant capacity on STZ-induced calpain/Cdk5/Drp1 activation.(A) Intracellular ROS was observed in STZ (24 h)-treated HT-22 and Prx1-expressed cells with CM-H 2 DCFDA (5 μM) staining using fluorescent microscopy; scale bar, 200 μm.(B) Intracellular ROS level was analyzed in STZ (24 h)-treated HT-22 and Prx1-expressed HT-22 cells using flow cytometry with CM-H 2 DCFDA staining.(C) Calpain-2 protein expression level in STZ (1.5h)-treated HT-22 cells pretreated with or without NAC (5 mM) were confirmed with western blotting analysis.(D) p25/35 and Cdk5 proteins expression level in STZ (6 h)-treated HT-22 cells pretreated with or without NAC were confirmed with western blotting analysis.(E) p-Drp1 (S616), (F) Mitochondrial Drp1 levels in STZ (12 h)-treated HT-22 cells pretreated with or without NAC were confirmed with western blotting.COXIV was used as the loading control for the mitochondria.(G) Change in mitochondrial morphology was observed using confocal microscopy in STZ (12 h)-treated DsRed2-mito expressed HT-22 cells pretreated with or without NAC.The bottom panels showed the magnified images of regions indicated by white squares in the top panels; scale bar, 5 μm.The graph showed distribution of all mitochondrial particles and average mitochondrial length.(H) Intracellular ATP levels were measured in STZ (12 h)-treated HT-22 cells pretreated with or without NAC.Blots were cropped to highlight the region of interest; full blot images in this article are provided in supplementary information.The data are presented as mean values ± SD (n ≥ 3).* denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Figure 7 .
Figure 7. Effect Prx1 antioxidant capacity on STZ-induced AD-like pathology.(A) Cleaved Caspase-3 and cleaved PARP, (B) NeuN, (C) PSD95, (D) p-Tau(S262) and AT8(S202/T205) protein expression level were confirmed by western blotting analysis in STZ-treated HT-22 cells pretreated with or without NAC.Blots were cropped to highlight the region of interest; full blot images in this article are provided in supplementary information.The data are presented as mean values ± SD (n ≥ 3).* denotes p < 0.05, and *** denotes p < 0.001.